Prophylactic Prednisolone Promotes AAV5 Hepatocyte Transduction Through the Novel Mechanism of AAV5 Coreceptor Platelet-Derived Growth Factor Receptor Alpha Upregulation and Innate Immune Suppression

Adeno-associated virus (AAV) vectors are used to deliver therapeutic transgenes, but host immune responses may interfere with transduction and transgene expression. We evaluated prophylactic corticosteroid treatment on AAV5-mediated expression in liver tissue. Wild-type C57BL/6 mice received 6 × 1013 vg/kg AAV5-HLP-hA1AT, an AAV5 vector carrying a human α1-antitrypsin (hA1AT) gene with a hepatocyte-specific promoter. Mice received 4 weeks of daily 2 mg/kg prednisolone or water starting day −1 or 0 before vector dosing. Mice that received prophylactic corticosteroids had significantly higher serum hA1AT protein than mice that did not, starting at 6 weeks and persisting to the study end at 12 weeks, potentially through a decrease in the number of low responders. RNAseq and proteomic analyses investigating mechanisms mediating the improvement of transgene expression found that prophylactic corticosteroid treatment upregulated the AAV5 coreceptor platelet-derived growth factor receptor alpha (PDGFRα) on hepatocytes and downregulated its competitive ligand PDGFα, thus increasing the uptake of AAV5 vectors. Evidently, prophylactic corticosteroid treatment also suppressed acute immune responses to AAV. Together, these mechanisms resulted in increased uptake and preservation of the transgene, allowing more vector genomes to be available to assemble into stable, full-length structures mediating long-term transgene expression. Prophylactic corticosteroids represent a potential actionable strategy to improve AAV5-mediated transgene expression and decrease intersubject variability.


INTRODUCTION
][7][8][9][10][11] Host cell pattern recognition receptors induce proinflammatory cytokines, and antiviral responses lead to vector deoxyribonucleic acid (DNA) degradation. 5,12Adaptive immune responses, including expansion of CD8 + T cells directed against transgene-expressing target cells, cause inflammation and damage. 7,13,14orticosteroids inhibit immune responses by suppressing transcription of proinflammatory cytokines and chemokines. 15,16Corticosteroids are frequently used with AAV gene therapies to treat alanine aminotransferase (ALT) elevations in liver-targeted gene therapies, and preserve transgene expression in some cases. 8,10][19] Prophylactic corticosteroids administered before AAV dosing may promote transgene expression.In mice, prophylactic dexamethasone administered 2 h before AAV9 dosing increased hepatic levels of vector genomes. 20onhuman primates (NHPs) treated with prophylactic prednisolone before AAV dosing had less CD8 + T cell infiltration and apoptosis than those who did not. 21However, prednisolone treatment in mice 1 week after AAV5-hFVIII-SQ dosing did not affect transgene expression. 22imilarly, dexamethasone treatment initiated 1 year after AAV9 did not change transgene expression levels in dogs. 20n this study, we assessed transgene expression in mice after prophylactic prednisolone before AAV5-mediated gene therapy over 12 weeks.After confirming prophylactic prednisolone increased transgene expression, we investigated potential mechanisms of action at 2 and 24 h post-AAV dosing using RNAseq, proteomics, and targeted molecular analyses.Not only did prophylactic prednisolone suppress innate immune responses, it also upregulated the expression of the AAV5 coreceptor platelet-derived growth factor receptor alpha (PDGFRa).

Study design
We assessed the effects of prophylactic steroids over 12 weeks with wild-type C57BL/6 mice ( Jackson Laboratory, Bar Harbor, ME) that received either water or 2 mg/kg prednisolone in water vehicle by oral gavage beginning on day -1 or 0 before AAV dosing and continuing for 4 weeks (Fig. 1A).Blood was drawn at baseline and weeks 1, 4, 6, 8, and 12 postdose for serum hA1AT measurement; takedown cohorts were collected at weeks 4 (n = 3) and 12 (n = 7) and livers were collected for vector genome quantification.Mice were dosed with 6 • 10 13 vg/kg AAV5-HLP-hA1AT.The impact of prophylactic methylprednisolone and rituximab before dosing with 6 • 10 13 vg/kg AAV5-hFVIII-SQ was assessed in NHPs (Supplementary Methods section in Supplementary Data).
We also assessed the effects of prophylactic steroids on events immediately after AAV dosing with wild-type C57BL/6 mice who received one of four treatments (prophylactic prednisolone or water and 6 • 10 13 vg/kg AAV5-HLP-hA1AT or vehicle) before euthanization at 2 or 24 h post-AAV dose (Fig. 2A).Blood samples were taken predose and terminally for hA1AT and other protein assays.Livers were collected for RNAseq, protein biomarker detection using an Olink assay, N-glycome analyses, and immunohistochemistry.
In addition, we assessed how natural PDGFRa variation correlates with transgene expression by dosing wildtype C57BL/6 and BalbC ( Jackson Laboratory) mice with 6 • 10 13 AAV5-hFVIII-SQ.Mice were euthanized at 5 weeks postdose, and blood and liver samples were collected for assessment of FVIII-SQ protein, transgene DNA, and PDGFRa protein.

Ethics statement
Mouse protocols were approved by the Animal Resource Committee of BioMarin Pharmaceutical, Inc. and the Institutional Animal Care and Use Committee of the Buck Institute.NHP protocols were approved by the Institutional Animal Care and Use Committee at the Charles River Laboratory (San Francisco, CA).

RNAseq and pathway enrichment analyses
mRNA samples were prepared using Illumina Stranded mRNA kits (Illumina, San Diego, CA) and sequenced on a NovaSeq 6000 v1.5 using single-end 100-bp reads.Reads were analyzed and mapped to the GRCm38/mm10 mouse genome assembly. 27Differential expression was determined using the edgeR package in R (R Foundation, Vienna, Austria).Gene set enrichment analysis was performed using hallmark gene sets from MSigDb in R, using normalized enrichment score (NES) and Benjamini-Hochberg-adjusted p value. 28,29otein assays hA1AT protein in mice was measured in serum (12-week study) or plasma (24-h study) using an enzyme-linked immunosorbent assay (ELISA) detecting hA1AT without mouse cross-reactivity (Human Serpin A1 PROPHYLACTIC PREDNISONE IMPROVES AAV TRANSDUCTION DuoSet ELISA, DY1268; R&D Systems, Minneapolis, MN; Supplementary Methods section in Supplementary Data).hFVIII-SQ protein was measured in plasma samples using a sandwich ELISA utilizing human-specific anti-FVIII capture (Green Mountain Antibodies, Burlington, VT) and detection (F8C-EIA; Affinity Biologics, Ancaster, ON, Canada) antibody pairs, as previously described. 23omplement system activity was assessed with plasma C3b (Supplementary Methods section in Supplementary Data).An Olink proteomic assay was used to explore protein expression (Supplementary Methods section in Supplementary Data).Plasma PDGFa/a was measured using a Mice PDGFa/a ELISA kit (Abcam, Cambridge, United Kingdom).

Plasma ALT
Mouse plasma ALT levels were assessed at 2 and 24 h post-AAV dose and over 12 weeks (Supplementary Methods section in Supplementary Data).

DNA in situ hybridization
Hepatocytes staining positive for vector genome and in situ hybridization (ISH) area per cell were quantified in formalin-fixed paraffin-embedded (FFPE) liver sections of 5 lm prepared as described. 23One whole liver section was acquired per animal, and two regions were randomly selected for analysis using Visiopharm (Hørsholm, Denmark).

Droplet digital polymerase chain reaction
Vector genome quantification, including full-length genomes and overall total genomes, was performed with drop-phase droplet digital polymerase chain reaction (ddPCR) analyses targeting the AAV5-hA1AT-HLP transgene sequence, as described 23 (Supplementary Methods section in Supplementary Data), following DNA and ribonucleic acid (RNA) extraction using the AllPrep DNA/RNA Mini kit (Qiagen, Hilden, Germany).

N-glycome analysis
Changes in N-glycosylation in mouse livers at 2 and 24 h post-AAV dose were assessed (Supplementary Methods section in Supplementary Data).• magnification.For (B), a Welch's unpaired t-test was performed.For plasma PDGFRa in (C) a one-way ANOVA followed by Tukey's multiple comparison showed no significant differences.

Immunohistochemistry
Hepatic expression and distribution of PDGFRa and viral protein 3 (VP3) were measured by immunohistochemistry. FFPE livers sectioned at 5-lm thickness were collected on Leica Microsystems Plus Slides (Leica Biosystems, Buffalo Grove, IL).Immunostaining was performed using a Leica BOND RX Autostainer (Supplementary Methods section in Supplementary Data).

hFVIII-SQ vector genome quantification
AAV5-hFVIII-SQ vector genome levels were measured with a quantitative real-time PCR assay using a TaqMan DNA probe and primers specific to hFVIII-SQ after genomic liver DNA was extracted using the Quick DNA/RNA MagBead kit (Zymo Research, Irvine, CA; Supplementary Methods section in Supplementary Data).

PDGFRa in vitro analyses
PDGFRa expression upregulation by prophylactic prednisolone was assessed in human and mouse primary hepatocytes in vitro (Supplementary Methods section in Supplementary Data).The effect of PDGFRa on AAV transduction was confirmed by knockdown in HepG2 cells with a short hairpin RNA (shRNA; Supplementary Methods section in Supplementary Data).

Statistical methods
Serum hA1AT was analyzed using a Student's t-test at each time point after log 10 transformation.Benjamini-Hochberg-adjusted p values were calculated for differential expression and gene set enrichment analyses in RNAseq experiments and Olink proteomic analysis using p < 0.05.Plasma FVIII protein and liver hFVIII-SQ vector genome copies were analyzed by a Student's t-test.Plasma PDGFa/ a, complement system activation, and immunohistochemistry staining intensity were analyzed using one-way analysis of variance (ANOVA) and a Tukey's multiple comparison test.PDGFRa and PDGFa normalized expression were compared using a Welch's unpaired t-test.

Prophylactic prednisolone treatment increases transgene expression and vector DNA
In the 12-week study, mice who received prophylactic prednisolone on either day -1 or 0 had significantly higher serum hA1AT protein than nonprednisolone-treated mice from week 6 onward (Supplementary Fig. S1A).Serum protein was *1.5to 2-fold higher for mice receiving prednisolone at either time point in weeks 6 through 12 than for nontreated mice; prophylactic prednisolone appeared to lower interindividual variability by reducing the number of mice with low expression (Fig. 1B).
More hepatocytes stained positive for vector DNA in mice that received prophylactic prednisolone versus water on either day -1 or 0 at weeks 4 and 12 (Supplementary Fig. S1B), although significance was not reached.A clear trend for more overall and full-length vector genomes was also detected in prednisolone-treated mice compared with nonprednisolone-treated mice (Fig. 1C and Supplementary Fig. S1C).Similarly, after dosing with AAV5-hFVIII-SQ, significantly more full-length vector DNA ( p < 0.05) was present in the livers of NHPs that received prophylactic methylprednisolone and rituximab than in those that did not (Supplementary Fig. S2).
In mice, serum hA1AT protein levels correlated significantly (r = 0.68, p < 0.0001) with full-length vector genomes in the liver at week 12 (Fig. 1D).Transgene RNA levels also correlated with vector DNA (r = 0.49, p < 0.01) and transgene protein (r = 0.59, p < 0.001) at 12 weeks (Supplementary Fig. S1D, E).Overall, prophylactic corticosteroids improved AAV5-mediated transgene expression by increasing levels of functional full-length vector DNA in hepatocytes, giving rise to more RNA and protein.

Prophylactic prednisolone promotes transduction within 24 h of AAV dosing
Previously, initiation of prednisolone 1 week after AAV5-hFVIII-SQ dosing did not modulate transgene expression, 22 while here, initiating prednisolone before AAV dosing resulted in improved transgene expression.Hence, we hypothesized that the mechanism promoting AAV-mediated expression in response to prophylactic prednisolone occurs early and investigated potential mechanisms active immediately after AAV and steroid dosing. 22Mice received a single treatment of prednisolone or water 2 h before dosing with AAV or vehicle (Fig. 2A), and blood and livers were collected at 2 and 24 h.

Prednisolone treatment suppresses innate immune responses
For mice treated with prophylactic prednisolone versus control before AAV dosing, 863 and 1561 genes were significantly upregulated and downregulated, respectively, at 2 h postdose.By 24 h, only 87 and 173 genes were upregulated and downregulated, respectively.Inflammatory response pathways, such as interferon-a and tumor necrosis factor-a/nuclear factor-jB signaling, were downregulated by prednisolone as indicated by NES (Table 2).We specifically examined IL-1b expression as a marker of the inflammasome and found it was significantly downregulated by prednisolone (Supplementary Fig. S3A).
We used an exploratory Olink mouse panel (Uppsala, Sweden) to measure the effect of prophylactic prednisolone on plasma proteins at 2 and 24 h post-AAV dose.Normalized protein expression of most inflammatory response proteins was significantly suppressed by prophylactic prednisolone at both time points ( p < 0.05; Supplementary Tables S2 and S3), but cytokines were upregulated by AAV treatment ( p < 0.05; Supplementary Tables S4 and S5).However, serum ALT levels indicating liver damage were not significantly elevated with or without prednisolone (Supplementary Fig. S4A, B).
In addition, despite RNAseq data showing complement activation by AAV in the liver (Supplementary Table S6), there were no significant differences in plasma C3b levels between treatments (Supplementary Fig. S3B).

Prednisolone treatment upregulates the AAV5 coreceptor PDGFRa
Because prophylactic prednisolone increased vector DNA, we hypothesized it enhances initial trans-duction efficacy.We thus investigated mechanisms involved in the initial uptake of AAV vectors.][32][33][34][35][36][37][38] Liver levels of N-glycans in mice at 2 and 24 h were not affected by prophylactic prednisolone (Supplementary Fig. S5).In our RNAseq data set, PDGFRa expression at 2 h post-AAV dose was significantly higher in livers of mice that received prednisolone versus water before AAV treatment, and PDGFa, a ligand for PDGFR, was significantly lower in mice that received prednisolone versus NES determines whether a gene set is moving up (positively regulated) or down (negatively regulated) the gene rankings when comparing mice treated with prednisolone+AAV5-HLP-hA1AT versus mice treated with prednisolone+vehicle (n = 10/group).These curated data were sorted by NES.
water before AAV dosing (Fig. 2B).However, differences did not persist to 24 h.AAVR or PDGFRb expression did not significantly differ (data not shown).
Secreted PDGFa forms homodimer (PDGFa/a) or PDGFb heterodimer complexes (PDGFa/b), and so, to confirm RNAseq data, circulating ligands were assessed using an ELISA for PDGFa/a and an Olink assay for PDGFb/b (both also detect PDGFa/b).At 2 h post-AAV, plasma levels of PDGF ligands were lower, although not significant, in mice that received prophylactic prednisolone versus vehicle (Fig. 2C).Mass spectrometry of total liver homogenate samples found no significant difference between groups in PDGFRa (data not shown).As liver homogenate may include lysates multiple cell types that could dilute signals, we also used immunohistochemistry to detect PDGFRa expression on hepatocytes.Analysis confirmed a significantly higher PDGFRa protein in a subset of hepatocytes at 2 h post-AAV dosing in mice treated with prophylactic prednisolone versus vehicle (Fig. 2D).
In addition, prophylactic prednisolone in mice increased transgene DNA per cell as measured by ddPCR in the liver homogenate (Fig. 3D); however, these differences did not reach statistical significance.At the 2-and 24-h time points, the ISH signals for vector DNA were saturating, rendering quantitative analyses unfeasible.Therefore, one additional cohort was generated and taken down 1 week post-AAV dosing for ISH.The mean percentage of hepatocytes stained positive for vector DNA was significantly higher in mice treated with prophylactic prednisolone (79.2%) versus control (63.2%;Fig. 3E).Increased liver vector genomes resulted from both a higher percentage of hepatocytes taking up vector DNA and more uptake per hepatocyte (Fig. 3E).These results suggest that acute upregulation of PDGRFa proximal to AAV dosing increases levels of vector DNA within hepatocytes.

PDGFRa expression variation may contribute to intersubject variability
We assessed potential contributions of individual natural variation in PDGFRa expression to variable transgene expression by comparing outcomes after AAV5-hFVIII-SQ dosing in C5BL/6 and BalbC mice.In C57BL/6 mice naive to AAV or corticosteroid treatment, endogenous PDGFRa liver expression is highly variable (Fig. 4A); after AAV5-hFVIII-SQ dosing, the percentage of hepatocytes stained positive for PDGFRa significantly correlated with those positive for vector genome DNA (Fig. 4B).

In vitro results confirm prednisolone increases PDGFRa expression, affecting AAV5 transduction
To assess if prednisolone affects PDGFRa expression in human hepatocytes, we treated primary hepatocytes with prednisolone.PDGRFa transcripts significantly increased in both human and murine hepatocytes (Fig. 4G), as well as monkey and dog hepatocytes (data not shown).Furthermore, shRNA knockdown of PDGFRa in the human hepatocyte HepG2 cell line significantly decreased AAV5 transduction (Fig. 4H).Altogether, prophylactic prednisolone increases PDGFRa expression, resulting in increased transduction of AAV5 vectors, leading to more vector DNA in hepatocytes, and improved transgene expression.

DISCUSSION
We investigated whether prophylactic prednisolone before AAV administration affected transgene expression.In mice, prophylactic prednisolone before AAV vector dosing increased steady-state transgene serum protein levels over 12 weeks and appeared to reduce interindividual variability in transgene protein expression.In mice and NHPs, prophylactic corticosteroids increased vector genome copy numbers in the liver.We then investigated mechanisms of improved transgene expression focusing on time points proximal to AAV dosing, since AAV uptake and acute innate immune responses occur rapidly following AAV administration. 41Overall, prophylactic prednisolone increased the uptake of AAV5 vectors by suppressing innate immune responses and upregulating the expression of the AAV5 receptor PDGFRa.Together, these mechanisms increased vector genomes available to assemble into stable, full-length structures mediating long-term transgene expression.
Surprisingly, prophylactic corticosteroids also modulated cellular mechanisms involved in receptor-mediated endocytosis of AAV5; this may explain why corticosteroids administered 1 week post-AAV have little effect. 22][64] Knockdown of PDGFRa expression resulted in lower vector DNA levels in transduced hepatocytes in vitro.This suggests that prophylactic prednisolone increases the expression of the AAV5 coreceptor PDGFRa and decrea-ses the expression of the competitive PDGF ligand complexes, resulting in an overall higher AAV5 uptake by hepatocytes.A significantly higher PDGFRa protein expression was present in hepatocytes 2 h after AAV dosing in mice treated with prophylactic prednisolone, coinciding with rapid cellular uptake of AAV vectors postdosing. 39,40ollectively, these data indicate that variation in AAV5 receptors, particularly PDGFRa, may be a host factor contributing to the variable response seen in gene therapy trials.Although PDGFRa expression is variable in human livers (Supplementary Fig. S6), its contribution to response differences in clinical trials is unknown. 65Insights will be provided by the ongoing phase 3 trial evaluating valoctocogene roxaparvovec with prophylactic corticosteroids in participants with hemophilia A (NCT04323098).A limitation of this research is the use of a mouse model for human disease, given immune system differences. 66e also did not consider the effect of vector dose, adaptive immunity, or mechanisms after capsid uptake.
Overall, prophylactic corticosteroids before dosing with AAV5-based gene therapy increase transgene expression in mice through multiple complementary mechanisms, including innate immune suppression and increased AAV5 uptake by hepatocytes.Therefore, prophylactic corticosteroids may represent an actionable strategy for increasing transgene expression and reducing interindividual variability in response to AAV5-mediated gene therapy.

Figure 2 .
Figure 2. Effect of prophylactic prednisolone treatment on PDGFRa receptor and ligands at 2 and 24 h after vector dosing.(A) Study design of the 2-and 24-h study.(B) Normalized expression of PDGFRa and

Figure 3 .
Figure 3. Upregulation of PDGFRa expression in hepatocytes correlates with increased AAV5 transduction.(A) AAV5 uptake into hepatocytes 2 h following AAV5-HLP-hA1AT administration with and without prophylactic prednisolone treatment as indicated with VP3.(B) PDGFRa and AAV5 uptake (via VP3) coincide in the same hepatocytes 2 h following AAV5-HLP-hA1AT administration with and without prophylactic prednisolone treatment.(C) Pearson correlation of PDGFRa and VP3 intensity in hepatocytes 2 h following AAV5-HLP-hA1AT administration with and without prophylactic prednisolone treatment.(D) ddPCR

Table 1 .
Enrichment of hallmark gene sets for the effect of adeno-associated virus at 2 and 24 hours postdose

Table 2 .
Enrichment of hallmark gene sets for the effect of prophylactic prednisolone at 2 and 24 hours postdosing with adeno-associated virus